RESUMEN

Acute upper respiratory tract infections (URTIs) are caused by numerous viruses and bacteria. URTIs can be a cause of morbidity and are among the most common reasons for visiting healthcare practitioners and prescribing antibiotics to children in addition to causing absenteeism from school and work. Oral intake of Lacticaseibacillus rhamnosus GG DSM 33156 has shown beneficial health effects in several clinical trials, primarily relating to immune function and gastrointestinal health in children and adults. It has also been suggested that oral intake of L. rhamnosus GG DSM 33156 can reduce the incidence rate and alleviate symptoms of URTIs in children. We here report the results of a randomised, double-blind, placebo-controlled trial of 619 children aged 2-6 years conducted at a single centre in Scotland. The children, who were in day care or primary school, were followed over a 16-week intervention period with 309 randomised in the active group and 310 in the placebo group. The parents or guardians reported a daily healthcare status and any presumed episodes of URTI, which were subsequently confirmed by a general practitioner. The investigational product was well tolerated in the trial. Although a general trend towards a beneficial effect was observed, this trial did not demonstrate that L. rhamnosus GG DSM 33156 significantly reduced the incidence of URTIs, diagnosed by a general practitioner according to prespecified criteria (primary endpoint). Moreover, none of the secondary efficacy endpoints were met. Applying a Ward's hierarchical clustering, two separate clusters, focussing on four quality of life-related endpoints, were identified. Cluster 1 was associated with more severe URTI characteristics than cluster 2. Cluster 2 was significantly enriched with children who consumed the product, indicating that the symptoms children experience during an URTI are alleviated by the intake of L. rhamnosus GG DSM 33156. The study is registered at ClinicalTrials.gov ID: NCT03636191.

Asunto(s)

Lacticaseibacillus rhamnosus , Probióticos , Infecciones del Sistema Respiratorio , Adulto , Niño , Preescolar , Método Doble Ciego , Humanos , Probióticos/uso terapéutico , Calidad de Vida , Sistema Respiratorio , Infecciones del Sistema Respiratorio/tratamiento farmacológico

RESUMEN

Multicolour fluorescence imaging by STimulated Emission Depletion (STED) superresolution microscopy with doughnut-shaped STED laser beams based on different wavelengths for each colour channel requires precise image registration. This is especially important when STED imaging is used for co-localisation studies of two or more native proteins in biological specimens to analyse nanometric subcellular spatial arrangements. We developed a robust postprocessing image registration protocol, with the aim to verify and ultimately optimise multicolour STED image quality. Importantly, this protocol will support any subsequent quantitative localisation analysis at nanometric scales. Henceforth, using an approach that registers each colour channel present during STED imaging individually, this protocol reliably corrects for optical aberrations and inadvertent sample drift. To achieve the latter goal, the protocol combines the experimental sample information, from corresponding STED and confocal images using the same optical beam path and setup, with that of an independent calibration sample. As a result, image registration is based on a strategy that maximises the cross-correlation between sequentially acquired images of the experimental sample, which are strategically combined by the protocol. We demonstrate the general applicability of the image registration protocol by co-staining of the ryanodine receptor calcium release channel in primary mouse cardiomyocytes. To validate this new approach, we identify user-friendly criteria, which - if fulfilled - support optimal image registration. In summary, we introduce a new method for image registration and rationally based postprocessing steps through a highly standardised protocol for multicolour STED imaging, which directly supports the reproducibility of protein co-localisation analyses. Although the reference protocol is discussed exemplarily for two-colour STED imaging, it can be readily expanded to three or more colours and STED channels.

Asunto(s)

Procesamiento de Imagen Asistido por Computador/métodos , Miocitos Cardíacos/enzimología , Imagen Óptica/métodos , Canal Liberador de Calcio Receptor de Rianodina/análisis , Animales , Células Cultivadas , Ratones

RESUMEN

PURPOSE: Congenital disorders of glycosylation (CDG) result from mutations in N-glycan biosynthesis. Mutations in phosphomannomutase (PMM2) cause CDG-Ia. Here, we report four clinically mild patients and their mutations in PMM2. METHODS: Analysis of the PMM2 cDNA and gene revealed the mutations affecting the glycosylation efficiency. RESULTS: The patients have 30% to 50% normal PMM activity in fibroblasts due to different mutations in PMM2, and we studied the effect of each mutation on the PMM activity in a Saccharomyces cerevisiae expression system. CONCLUSIONS: Each patient carried a severe mutation that decreased the PMM activity to less than 10% as well as a relatively mild mutation. A new mutation, deletion of base 24, changed the reading frame. The C9Y, C241S, and L32R mutations showed 27% to 45% activity when expressed in the eukaryotic expression system, and the more severe D148N was shown to be thermolabile.

Asunto(s)

Trastornos Congénitos de Glicosilación/enzimología , Trastornos Congénitos de Glicosilación/genética , Mutación , Fosfotransferasas (Fosfomutasas)/genética , Alelos , Niño , Preescolar , Trastornos Congénitos de Glicosilación/diagnóstico , Análisis Mutacional de ADN , Femenino , Fibroblastos/enzimología , Fibroblastos/metabolismo , Genotipo , Glicosilación , Humanos , Masculino , Núcleo Familiar , Linaje , Fenotipo , Fosfotransferasas (Fosfomutasas)/metabolismo , Sistemas de Lectura , Saccharomyces cerevisiae/genética , Piel/citología

RESUMEN

Complete loss of N-glycosylation is lethal in both yeast and mammals. Substantial deficiencies in some rate-limiting biosynthetic steps cause human congenital disorders of glycosylation (CDG). Patients have a range of clinical problems including variable degrees of mental retardation, liver dysfunction, and intestinal disorders. Over 60 mutations in phosphomannomutase (encoded by PMM2) diminish activity and cause CDG-Ia. The severe mutation R141H in PMM2 is lethal when homozygous, but heterozygous in about 1/70 Northern Europeans. Another disorder, CDG-Ic, is caused by mutations in ALG6, an alpha 1,3glucosyl transferase used for lipid-linked precursor synthesis, yet some function-compromising mutations occur at a high frequency in this gene also. Maintenance of seemingly deleterious mutations implies a selective advantage or positive heterosis. One possible explanation for this is that production of infective viruses such as hepatitis virus B and C, or others that rely heavily on host N-glycosylation, is substantially inhibited when only a tiny fraction of their coat proteins is misglycosylated. In contrast, this reduced glycosylation does not affect the host. Prevalent functional mutations in rate-limiting glycosylation steps could provide some resistance to viral infections, but the cost of this insurance is CDG. A balanced glycosylation level attempts to accommodate these competing agendas. By assessing the occurrence of a series of N-glycosylation-compromising alleles in multi-genic diseases, it may be possible to determine whether impaired glycosylation is a risk factor or a major determinant underlying their pathology.

Asunto(s)

Errores Innatos del Metabolismo de los Carbohidratos/metabolismo , Glicosilación , Animales , Errores Innatos del Metabolismo de los Carbohidratos/genética , Humanos , Mutación , Factores de Riesgo

RESUMEN

BACKGROUND: Recent advances in high-speed scanning technology have enabled a new generation of optical coherence tomographic (OCT) systems to perform imaging at video rate. Here, a handheld OCT probe capable of imaging the anterior segment of the eye at high frame rates is demonstrated for the first time. OBJECTIVE: To demonstrate real-time OCT imaging of anterior segment structures. DESIGN: Survey of anterior segment structures in normal human subjects. SETTING: Laboratory. MAIN OUTCOME MEASURES: Achieving real-time imaging of the anterior segment, satisfactory image quality, and convenience of a handheld probe. RESULTS: Optical coherence tomographic imaging of the anterior segment of the eyes of human subjects was performed using 1310-nm wavelength light with an image rate of 8 frames per second. Imaging trials demonstrated clear resolution of corneal epithelium and stroma, sclerocorneal junction, sclera, iris pigment epithelium and stroma, and anterior lens capsule. The anterior chamber angle was clearly visualized. Limited imaging of the ciliary body was performed. Real-time imaging of pupillary constriction in response to light stimulus was also performed. CONCLUSION: High-speed OCT at 1310-nm wavelength is a potentially useful technique for noninvasive assessment of anterior segment structures. CLINICAL RELEVANCE: Our results suggest that real-time OCT has potential applications in glaucoma evaluation and refractive surgery.

Asunto(s)

Segmento Anterior del Ojo/anatomía & histología , Técnicas de Diagnóstico Oftalmológico/instrumentación , Procesamiento de Imagen Asistido por Computador/instrumentación , Cámara Anterior/anatomía & histología , Cuerpo Ciliar/anatomía & histología , Sistemas de Computación , Humanos , Interferometría/instrumentación , Iris/anatomía & histología , Cristalino/anatomía & histología , Luz , Músculo Liso/anatomía & histología , Esclerótica/anatomía & histología , Tomografía

RESUMEN

BACKGROUND: Both optical coherence tomography (OCT) and catheter probe EUS (CPEUS) are candidates for high-resolution imaging of the GI wall, but their potential roles in this clinical context have not been investigated. METHODS: OCT and CPEUS were used to image normal-appearing portions of the GI tract at the same sites. CPEUS was performed with a 20-MHz or a new 30-MHz catheter probe. RESULTS: Forty-four histologically confirmed normal sites in 27 patients were evaluated. With OCT, mucosa and muscularis mucosa were clearly seen at all sites. Except for stomach, OCT demonstrated the submucosa in all sites. OCT penetration ranged from 0.7 to 0.9 mm. Microscopic structures such as esophageal glands, intestinal villi, colonic crypts, and blood vessels were easily identified. CPEUS penetration ranged from 10 mm to 20 mm, and 5 to 7 distinct layers were discernible. However, both mucosa and submucosa were seen as thin layers without microscopic detail. CONCLUSION: OCT resolution is superior to high-frequency CPEUS, but depth of penetration is limited to mucosa and submucosa. OCT images the major structural components of the mucosa and submucosa whereas CPEUS does not. Potentially, OCT and high-frequency CPEUS may be complementary for clinical imaging.

Asunto(s)

Endoscopía Gastrointestinal/métodos , Endosonografía/métodos , Tomografía/métodos , Humanos

RESUMEN

Optical-thermal models that can accurately predict temperature rise and damage in blood vessels and surrounding tissue may be used to improve the treatment of vascular disorders. Verification of these models has been hampered by the lack of time- and depth-resolved experimental data. In this preliminary study, an optical coherence tomography system operating at 4-30 frames per second was used to visualize laser irradiation of cutaneous (hamster dorsal skin flap) blood vessels. An argon laser was utilized with the following parameters: pulse duration 0.1-2.0 s, spot size 0.1-1.0 mm, power 100-400 mW. Video microscopy images were obtained before and after irradiations, and optical-thermal modelling was performed on two irradiation cases. Time-resolved optical coherence tomography and still images were compared with predictions of temperature rise and damage using Monte Carlo and finite difference techniques. In general, predicted damage agreed with the actual blood vessel and surrounding tissue coagulation seen in images. However, limitations of current optical-thermal models were identified, such as the inability to model the dynamic changes in blood vessel diameter that were seen in the optical coherence tomography images.

Asunto(s)

Vasos Sanguíneos/anatomía & histología , Rayos Láser , Tomografía/instrumentación , Tomografía/métodos , Animales , Argón , Vasos Sanguíneos/metabolismo , Cricetinae , Calor , Microscopía por Video , Modelos Teóricos , Método de Montecarlo , Temperatura , Factores de Tiempo , Enfermedades Vasculares/terapia

RESUMEN

Congenital disorders of glycosylation (CDG) are caused by autosomal recessive mutations in genes affecting N-glycan biosynthesis. Mutations in the PMM2 gene, which encodes the enzyme phosphomannomutase (mannose 6-phosphate <--> mannose 1-phosphate), give rise to the most common form: CDG-Ia. These patients typically present with dysmorphic features and neurological abnormalities, cerebellar hypoplasia, ataxia, hypotonia, and coagulopathy, in addition to feeding problems. However, the clinical symptoms vary greatly. The great majority of known CDG-Ia patients are of European descent where the most common mutant alleles originated. This ethnic bias can also be explained by lack of global awareness of the disorder. Here we report an Asian patient with prominent systemic features that we diagnosed with CDG-Ia resulting from two new mutations in the PMM2 gene (310C --> G resulting in L104V and an intronic mutation IVS1-1G --> A). The latter mutation seems to result in lower mRNA levels, and the L104V has been functionally analyzed in a yeast expression system together with known mutations. The Filipino and Cambodian origins of the parents show that CDG-Ia mutations occur in these ethnic groups as well as in Caucasians.

Asunto(s)

Trastornos Congénitos de Glicosilación/genética , Fosfotransferasas (Fosfomutasas)/genética , Secuencia de Aminoácidos , Asiático , Cambodia/etnología , Trastornos Congénitos de Glicosilación/enzimología , Trastornos Congénitos de Glicosilación/patología , Análisis Mutacional de ADN , ADN Complementario/química , ADN Complementario/genética , Salud de la Familia , Femenino , Prueba de Complementación Genética , Humanos , Lactante , Mutación , Filipinas/etnología , Fosfotransferasas (Fosfomutasas)/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Homología de Secuencia de Aminoácido

RESUMEN

We report the diagnosis and follow-up of two sibs reported in 1980 with recurrent venous thromboses and protein-losing enteropathy; one sib with biopsy-proven hepatic fibrosis died at age 5. The combination of symptoms was suggestive of the recently characterized congenital disorder of glycosylation type Ib (CDG-Ib), which is caused by a deficiency of the enzyme phosphomannose isomerase (PMI). An abnormal serum transferrin isoelectric focusing (IEF) pattern and a reduced PMI activity confirmed the diagnosis of CDG-Ib. Furthermore, mutational analysis of the MPI gene revealed two missense mutations, 419 T --> C (I140T) and 636 G --> A (R219Q), a single base substitution in intron 5, 670 + 9G --> A, as well as a polymorphism 1131A --> C (V377V) in both sibs. The surviving 33-year-old sib has had no further symptoms following childhood. Short-term low-dose oral mannose supplementation improved her transferrin IEF pattern and normalized her antithrombin III activity, further substantiating the beneficial effect of mannose in CDG-Ib. When her mannose blood level was measured, she showed a lower steady-state level but a faster mannose clearance rate. These results suggest that the clinical manifestations of PMI deficiency, although serious in childhood, can improve with age, even without mannose therapy, and allow for a normal adult life. However, the long-term prognosis may vary from patient to patient.

Asunto(s)

Trastornos Congénitos de Glicosilación/genética , Manosa-6-Fosfato Isomerasa/genética , Adulto , Preescolar , Trastornos Congénitos de Glicosilación/enzimología , Trastornos Congénitos de Glicosilación/patología , ADN/química , ADN/genética , Análisis Mutacional de ADN , ADN Complementario/química , ADN Complementario/genética , Salud de la Familia , Femenino , Estudios de Seguimiento , Humanos , Masculino , Manosa/administración & dosificación , Manosa/sangre , Manosa-6-Fosfato Isomerasa/deficiencia , Mutación Missense , Polimorfismo Genético , Factores de Tiempo , Transferrina/efectos de los fármacos , Transferrina/metabolismo , Resultado del Tratamiento

RESUMEN

We recently showed that a class of novel carboxylated N:-glycans was constitutively expressed on endothelial cells. Activated, but not resting, neutrophils expressed binding sites for the novel glycans. We also showed that a mAb against these novel glycans (mAbGB3.1) inhibited leukocyte extravasation in a murine model of peritoneal inflammation. To identify molecules that mediated these interactions, we isolated binding proteins from bovine lung by their differential affinity for carboxylated or neutralized glycans. Two leukocyte calcium-binding proteins that bound in a carboxylate-dependent manner were identified as S100A8 and annexin I. An intact N terminus of annexin I and heteromeric assembly of S100A8 with S100A9 (another member of the S100 family) appeared necessary for this interaction. A mAb to S100A9 blocked neutrophil binding to immobilized carboxylated glycans. Purified human S100A8/A9 complex and recombinant human annexin I showed carboxylate-dependent binding to immobilized bovine lung carboxylated glycans and recognized a subset of mannose-labeled endothelial glycoproteins immunoprecipitated by mAbGB3.1. Saturable binding of S100A8/A9 complex to endothelial cells was also blocked by mAbGB3.1. These results suggest that the carboxylated glycans play important roles in leukocyte trafficking by interacting with proteins known to modulate extravasation.

Asunto(s)

Ácidos Carboxílicos/metabolismo , Proteínas Portadoras/metabolismo , Movimiento Celular , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Leucocitos/metabolismo , Polisacáridos/metabolismo , Secuencia de Aminoácidos , Animales , Anexina A1/química , Anexina A1/inmunología , Anexina A1/metabolismo , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/farmacología , Antígenos de Diferenciación/inmunología , Antígenos de Diferenciación/aislamiento & purificación , Antígenos de Diferenciación/metabolismo , Antígenos de Diferenciación/fisiología , Sitios de Unión de Anticuerpos , Unión Competitiva/inmunología , Proteínas de Unión al Calcio/inmunología , Proteínas de Unión al Calcio/aislamiento & purificación , Proteínas de Unión al Calcio/metabolismo , Proteínas de Unión al Calcio/fisiología , Calgranulina A , Calgranulina B , Proteínas Portadoras/aislamiento & purificación , Proteínas Portadoras/fisiología , Bovinos , Adhesión Celular/inmunología , Membrana Celular/inmunología , Membrana Celular/metabolismo , Movimiento Celular/inmunología , Cromatografía de Afinidad/métodos , Endotelio Vascular/inmunología , Glicopéptidos/síntesis química , Glicopéptidos/metabolismo , Humanos , Sueros Inmunes/metabolismo , Sueros Inmunes/farmacología , Leucocitos/inmunología , Pulmón/citología , Pulmón/inmunología , Pulmón/metabolismo , Ratones , Datos de Secuencia Molecular , Peso Molecular , Neutrófilos/inmunología , Neutrófilos/metabolismo , Conejos , Proteínas S100/inmunología , Proteínas S100/aislamiento & purificación , Proteínas S100/metabolismo , Proteínas S100/fisiología , Homología de Secuencia de Aminoácido

RESUMEN

PDI1 is the essential gene encoding protein disulfide isomerase in yeast. The Saccharomyces cerevisiae genome, however, contains four other nonessential genes with homology to PDI1: MPD1, MPD2, EUG1, and EPS1. We have investigated the effects of simultaneous deletions of these genes. In several cases, we found that the ability of the PDI1 homologues to restore viability to a pdi1-deleted strain when overexpressed was dependent on the presence of low endogenous levels of one or more of the other homologues. This shows that the homologues are not functionally interchangeable. In fact, Mpd1p was the only homologue capable of carrying out all the essential functions of Pdi1p. Furthermore, the presence of endogenous homologues with a CXXC motif in the thioredoxin-like domain is required for suppression of a pdi1 deletion by EUG1 (which contains two CXXS active site motifs). This underlines the essentiality of protein disulfide isomerase-catalyzed oxidation. Most mutant combinations show defects in carboxypeptidase Y folding as well as in glycan modification. There are, however, no significant effects on ER-associated protein degradation in the various protein disulfide isomerase-deleted strains.

Asunto(s)

Retículo Endoplásmico/metabolismo , Escherichia coli/enzimología , Eliminación de Gen , Proteína Disulfuro Isomerasas/genética , Proteína Disulfuro Isomerasas/metabolismo , Pliegue de Proteína , Saccharomyces cerevisiae/enzimología , Western Blotting , Ditiotreitol/farmacología , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Genes Esenciales , Genes Fúngicos , Glicosilación , Mutación , Plásmidos/genética , Plásmidos/metabolismo , Pruebas de Precipitina , Proteína Disulfuro Isomerasas/química , Saccharomyces cerevisiae/genética

RESUMEN

INTRODUCTION: Outpatient knee arthroscopy under local analgesia can be performed solely as a diagnostic procedure. The aim was to estimate the diagnostic precision of such arthroscopy as compared to the diagnosis made during a secondary therapeutic operation, and to describe the flow of patients and the costs of this treatment strategy. MATERIAL AND METHODS: The records of 371 consecutive patients, who had a diagnostic knee arthroscopy performed under local analgesia, were reviewed retrospectively. The diagnosis made during the diagnostic arthroscopy (371 patients) and a later therapeutic operation (135 patients) were extracted and the patients were asked to fill in a questionnaire. RESULTS: The diagnostic arthroscopy could not be completed in 11 cases. No further operation was necessary in 188 patients. A secondary therapeutic operation was performed in 135 patients. In only 54% of these was the same diagnosis made during the diagnostic and the therapeutic operations. Only half of the 278 patients (75%) who returned the questionnaire, found that the diagnostic procedure had been pain free. DISCUSSION: With respect to the costs, the diagnostic arthroscopy cannot be recommended. Most economic was the strategy in which the diagnostic and therapeutic operations were performed together as an outpatient procedure. The relatively poor diagnostic precision of arthroscopy is surprising and should be kept in mind when patients continue to have unexplained complaints in the knee, despite a normal arthroscopy.

Asunto(s)

Artroscopía/métodos , Articulación de la Rodilla , Adulto , Procedimientos Quirúrgicos Ambulatorios/economía , Procedimientos Quirúrgicos Ambulatorios/métodos , Anestesia Local , Artroscopía/economía , Artroscopía/normas , Dinamarca , Femenino , Costos de la Atención en Salud , Humanos , Articulación de la Rodilla/patología , Articulación de la Rodilla/cirugía , Masculino , Persona de Mediana Edad , Pacientes Ambulatorios , Estudios Retrospectivos , Encuestas y Cuestionarios

RESUMEN

Intestinal biopsy in a boy with gastroenteritis-induced protein-losing enteropathy (PLE) showed loss of heparan sulfate (HS) and syndecan-1 core protein from the basolateral surface of the enterocytes, which improved after PLE subsided. Isoelectric focusing analysis of serum transferrin indicated a congenital disorder of glycosylation (CDG) and subsequent analysis showed three point mutations in the ALG6 gene encoding an alpha1,3-glucosyltransferase needed for the addition of the first glucose to the dolichol-linked oligosaccharide. The maternal mutation, C998T, causing an A333V substitution, has been shown to cause CDG-Ic, whereas the two paternal mutations, T391C (Y131H) and C924A (S308R) have not previously been reported. The mutations were tested for their ability to rescue faulty N:-linked glycosylation of carboxypeptidase Y in an ALG6-deficient Saccharomyces cerevisiae strain. Normal human ALG6 rescues glycosylation and A333V partially rescues, whereas the combined paternal mutations (Y131H and S308R) are ineffective. Underglycosylation resulting from each of these mutations is much more severe in rapidly dividing yeast. Similarly, incomplete protein glycosylation in the patient is most severe in rapidly dividing enterocytes during gastroenteritis-induced stress. Incomplete N:-linked glycosylation of an HS core protein and/or other biosynthetic enzymes may explain the selective localized loss of HS and PLE.

Asunto(s)

Enterocitos/metabolismo , Heparitina Sulfato/metabolismo , Mucosa Intestinal/metabolismo , Proteínas de la Membrana , Enteropatías Perdedoras de Proteínas/congénito , Enteropatías Perdedoras de Proteínas/metabolismo , Biopsia , Carboxipeptidasas/metabolismo , Fibroblastos/metabolismo , Glucosiltransferasas/deficiencia , Glicosilación , Histocitoquímica , Humanos , Inmunohistoquímica , Lactante , Mucosa Intestinal/patología , Masculino , Enteropatías Perdedoras de Proteínas/patología , Saccharomyces cerevisiae/metabolismo

RESUMEN

The PMM2 gene, which is defective in CDG-Ia, was cloned three years ago [Matthijs et al., 1997b]. Several publications list PMM2 mutations [Matthijs et al., 1997b, 1998; Kjaergaard et al., 1998, 1999; Bjursell et al., 1998, 2000; Imtiaz et al., 2000] and a few mutations have appeared in case reports or abstracts [Crosby et al., 1999; Kondo et al., 1999; Krasnewich et al., 1999; Mizugishi et al., 1999; Vuillaumier-Barrot et al., 1999, 2000b]. However, the number of molecularly characterized cases is steadily increasing and many new mutations may never make it to the literature. Therefore, we decided to collate data from six research and diagnostic laboratories that have committed themselves to a systematic search for PMM2 mutations. In total we list 58 different mutations found in 249 patients from 23 countries. We have also collected demographic data and registered the number of deceased patients. The documentation of the genotype-phenotype correlation is certainly valuable, but is out of the scope of this molecular update. The list of mutations will also be available online (URL: http://www.kuleuven. ac.be/med/cdg) and investigators are invited to submit new data to this PMM2 mutation database.

Asunto(s)

Trastornos Congénitos de Glicosilación/genética , Mutación Missense , Fosfotransferasas (Fosfomutasas)/genética , Adolescente , Adulto , Secuencia de Aminoácidos/genética , Niño , Trastornos Congénitos de Glicosilación/clasificación , Trastornos Congénitos de Glicosilación/enzimología , Trastornos Congénitos de Glicosilación/epidemiología , Exones/genética , Genotipo , Glicosilación , Humanos , Lactante , Recién Nacido , Datos de Secuencia Molecular , Fenotipo , Fosfotransferasas (Fosfomutasas)/metabolismo , Polimorfismo Genético/genética

RESUMEN

Congenital disorder of glycosylation Ic is caused by mutations in the hALG6 gene that encodes an alpha-1,3 glucosyltransferase. This enzyme is required for the addition of the first glucose residue to the lipid-linked oligosaccharide precursor for N-linked glycosylation. Here we describe the biochemical and molecular analysis of a patient with three mutations in the hALG6 gene. The maternal allele has an intronic G --> A mutation resulting in skipping of exon3 (IVS3 + 5G > A). This produces a nonfunctional enzyme as shown by its inability to restore normal glycosylation in a Saccharomyces cerevisiae strain lacking a functional ALG6. The paternal allele has two mutations. One is a deletion of three bases (895-897delATA) leading to an in-frame deletion of isoleucine 299 (delI299) located in a transmembrane domain. The second mutation on the same allele 911T > C causes a F304S change. When expressed in the ALG6 deficient yeast strain, this allele restores glycosylation but the mRNA is unstable or inefficiently transcribed, contributing to the impaired glycosylation in the patient.

Asunto(s)

Trastornos Congénitos de Glicosilación/genética , Glucosiltransferasas/genética , Proteínas de la Membrana , Mutación , Alelos , Niño , Trastornos Congénitos de Glicosilación/enzimología , Análisis Mutacional de ADN , Cartilla de ADN/química , Exones/genética , Fibroblastos/enzimología , Glicosilación , Humanos , Masculino , Polimorfismo Genético , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Saccharomyces cerevisiae/genética

RESUMEN

Congenital disorders of glycosylation (CDGs) are metabolic deficiencies in glycoprotein biosynthesis that usually cause severe mental and psychomotor retardation. Different forms of CDGs can be recognized by altered isoelectric focusing (IEF) patterns of serum transferrin (Tf). Two patients with these symptoms and similar abnormal Tf IEF patterns were analyzed by metabolic labeling of fibroblasts with ¿2-(3)Hmannose. The patients produced a truncated dolichol-linked precursor oligosaccharide with 5 mannose residues, instead of the normal precursor with 9 mannose residues. Addition of 250 microM mannose to the culture medium corrected the size of the truncated oligosaccharide. Microsomes from fibroblasts of these patients were approximately 95% deficient in dolichol-phosphate-mannose (Dol-P-Man) synthase activity, with an apparent K(m) for GDP-Man approximately 6-fold higher than normal. DPM1, the gene coding for the catalytic subunit of Dol-P-Man synthase, was altered in both patients. One patient had a point mutation, C(274)G, causing an R(92)G change in the coding sequence. The other patient also had the C(274)G mutation and a 13-bp deletion that presumably resulted in an unstable transcript. Defects in DPM1 define a new glycosylation disorder, CDG-Ie.

Asunto(s)

Trastornos Congénitos de Glicosilación/enzimología , Trastornos Congénitos de Glicosilación/genética , Manosiltransferasas/deficiencia , Manosiltransferasas/genética , Mutación , Encefalopatías Metabólicas Innatas/diagnóstico , Encefalopatías Metabólicas Innatas/enzimología , Encefalopatías Metabólicas Innatas/etiología , Secuencia de Carbohidratos , Células Cultivadas , Trastornos Congénitos de Glicosilación/complicaciones , Trastornos Congénitos de Glicosilación/diagnóstico , Análisis Mutacional de ADN , Discapacidades del Desarrollo/diagnóstico , Femenino , Fibroblastos/citología , Fibroblastos/enzimología , Glicósido Hidrolasas/metabolismo , Glicosilación , Humanos , Lactante , Focalización Isoeléctrica , Isoenzimas/deficiencia , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Manosa/metabolismo , Manosiltransferasas/metabolismo , Microcefalia/diagnóstico , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Convulsiones/diagnóstico , Eliminación de Secuencia , Transferrina/metabolismo

Asunto(s)

Trastornos Congénitos de Glicosilación/diagnóstico , Trastornos Congénitos de Glicosilación/terapia , Manosa/metabolismo , Trastornos Congénitos de Glicosilación/genética , Genes Recesivos/genética , Glicosilación , Humanos , Focalización Isoeléctrica/métodos , Manosa/uso terapéutico , Hipotonía Muscular/diagnóstico , Mutación/genética , Oligosacáridos/biosíntesis , Oligosacáridos/química , Trastornos Psicomotores/diagnóstico , Transferrina

RESUMEN

Protein folding catalysed by protein disulphide isomerase (PDI) has been studied both in vivo and in vitro using different assays. PDI contains a CGHC active site in each of its two catalytic domains (a and a'). The relative importance of each active site in PDI from Saccharomyces cerevisiae (yPDI) has been analysed by exchanging the active-site cysteine residues for serine residues. The activity of the mutant forms of yPDI was determined quantitatively by following the refolding of bovine pancreatic trypsin inhibitor in vitro. In this assay the activity of the wild-type yPDI is quite similar to that of human PDI, both in rearrangement and oxidation reactions. However, while the a domain active site of the human enzyme is more active than the a'-site, the reverse is the case for yPDI. This prompted us to set up an assay to investigate whether the situation would be different with a native yeast substrate, procarboxypeptidase Y. In this assay, however, the a' domain active site also appeared to be much more potent than the a-site. These results were unexpected, not only because of the difference with human PDI, but also because analysis of folding of procarboxypeptidase Y in vivo had shown the a-site to be most important. We furthermore show that the apparent difference between in vivo and in vitro activities is not due to catalytic contributions from the other PDI homologues found in yeast.

Asunto(s)

Carboxipeptidasas/química , Proteína Disulfuro Isomerasas/metabolismo , Saccharomyces cerevisiae/enzimología , Aprotinina/química , Aprotinina/metabolismo , Sitios de Unión , Carboxipeptidasas/metabolismo , Catálisis , Catepsina A , Disulfuros/química , Mutación , Oxidación-Reducción , Desnaturalización Proteica , Proteína Disulfuro Isomerasas/química , Proteína Disulfuro Isomerasas/genética , Proteína Disulfuro Isomerasas/aislamiento & purificación , Pliegue de Proteína , Saccharomyces cerevisiae/metabolismo

RESUMEN

Protein-disulfide isomerase (PDI) is an abundant folding catalyst in the endoplasmic reticulum of eukaryotic cells. PDI introduces disulfide bonds into newly synthesized proteins and catalyzes disulfide bond isomerizations. We have synthesized a library of disulfide-linked fluorescence-quenched peptides, individually linked to resin beads, for two purposes: 1) to probe PDI specificity, and 2) to identify simple, sensitive peptide substrates of PDI. Using this library, beads that became rapidly fluorescent by reduction by human PDI were selected. Amino acid sequencing of the bead-linked peptides revealed substantial similarities. Several of the peptides were synthesized in solution, and a quantitative characterization of pre-steady state kinetics was carried out. Interestingly, a greater than 10-fold difference in affinity toward PDI was seen for various substrates of identical length. As opposed to conventional PDI assays involving larger polypeptides, the starting material for this assay is homogenous. It is furthermore simple and highly sensitive (requires less than 0.5 microgram of PDI/assay) and thus opens the possibility for quantitative determination of PDI activity and specificity.

Asunto(s)

Oligopéptidos/metabolismo , Proteína Disulfuro Isomerasas/metabolismo , Secuencia de Aminoácidos , Catálisis , Disulfuros/metabolismo , Humanos , Cinética , Microscopía Fluorescente , Oligopéptidos/química , Oxidación-Reducción , Especificidad por Sustrato

RESUMEN

Protein disulphide isomerase is an enzyme that catalyses disulphide redox reactions in proteins. In this paper, fluorogenic and interchain disulphide bond containing peptide libraries and suitable substrates, useful in the study of protein disulphide isomerase, are described. In order to establish the chemistry required for the generation of a split-synthesis library, two substrates containing an interchain disulphide bond, a fluorescent probe and a quencher were synthesized. The library consists of a Cys residue flanked by randomized amino acid residues at both sides and the fluorescent Abz group at the amino terminal. All the 20 natural amino acids except Cys were employed. The library was linked to PEGA-beads via methionine so that the peptides could be selectively removed from the resin by cleavage with CNBr. A disulphide bridge was formed between the bead-linked library and a peptide containing the quenching chromophore (Tyr(NO2)) and Cys(pNpys) activated for reaction with a second thiol. The formation and cleavage of the interchain disulphide bonds in the library were monitored under a fluorescence microscope. Substrates to investigate the properties of protein disulphide isomerase in solution were also synthesized.

Asunto(s)

Disulfuros/química , Biblioteca de Péptidos , Proteína Disulfuro Isomerasas/metabolismo , Cistina/química , Colorantes Fluorescentes/metabolismo , Péptidos/síntesis química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Especificidad por Sustrato